Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• calcineurin inhibitor 1 in the preparation of a medicament for treating a disease associated with an increase in NF- ⁇ activity
• the present invention relates to the field of treatment of human tumors and inflammation, in particular to the inhibition of nuclear factor-kappaB (NF- ⁇ ) by calcineurin inhibitor 1 (RCAN1) protein and its use in the treatment of diseases associated with NF-KB, such as tumors.
• NF- ⁇ nuclear factor-kappaB
• RCAN1 calcineurin inhibitor 1
• Cancer is the leading cause of death in developed countries, and the latest data show that one in every four to five deaths in China is due to cancer. And most cancers still lack effective treatments, and cancer poses serious social and economic problems worldwide.
• NF- ⁇ nuclear factor -kappaB
• NF- ⁇ acts as a transcriptional regulator under normal physiological conditions and is primarily involved in immune responses, cell proliferation, cell death, and inflammatory responses (Baud and Karin, 2009).
• NF- ⁇ is the major anti-apoptotic factor in the body.
• the abnormality of NF-KB signaling pathway is related to the development of many tumors including blood and solid tumors and their tolerance to chemotherapy and radiotherapy. NF- ⁇ has been found.
• Hematological tumors with persistently abnormal increases include: multiple myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cells Lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome, adult T-cell leukemia, and solid tumors with abnormally high NF- ⁇ activity include: breast cancer, ovarian cancer, lung cancer, cervical cancer, prostate cancer, liver cancer , pancreatic cancer, esophageal cancer, gastric cancer, throat cancer, cholangiocarcinoma, thyroid cancer, parathyroid cancer, and squamous cell carcinoma of the skin and head and neck (Basseres and Baldwin, 2006).
• NF-KB promotes tumor development mainly through cell proliferation, angiogenesis, tumor metastasis, inflammatory response and apoptosis inhibition.
• the US FDA approved Bortezomib for the treatment of multiple myeloma As a proteasome inhibitor, bortezomib is thought to act primarily as a tumor by inhibiting the NF- ⁇ signaling pathway, but as A broad spectrum of proteasome inhibitors, bortezomib, has widespread and serious side effects that greatly limit its widespread use in cancer therapy (Delforge et al., 2010).
• Arsenic trioxide also known as arsenic
• its main role is also to inhibit NF-KB activity, but the toxicity of arsenic trioxide also limits its use in cancer therapy.
• NF-KB not only has important functions in tumors, but NF-KB is also a very important inflammatory factor, such as interleukin, tumor necrosis factor and other regulatory factors, and therefore has important functions in inflammatory reactions. Also, as an important molecule that affects cell survival, NF-KB also plays an important role in neurodegeneration, including dementia and stroke.
• the NF- ⁇ signaling pathway is a very important tumor drug target, and it is also a hot spot for drug development at home and abroad. There is an urgent need for drugs that can specifically inhibit NF- ⁇ activity and have fewer side effects for tumor therapy.
• Calcineurin inhibitor 1 was first discovered to be an endogenous inhibitor of calcineurin, and it has been found that the C-terminus of RCAN1 has calcineurin-inhibiting activity (Arron et al, 2006; Chan Et al, 2005; Fuentes et al, 2000). Due to the difference in the cleavage, RCAN1 has four splicing isoforms, wherein the cleavage isomer 1 is RCAN1.1 (SEQ ID NO: 1) and the cleavage isomer 4 is RCAN1.4 (SEQ ID NO) : 2) The expression in the body is higher.
• RCAN1.1 and RCAN1.4 differ only in the 28 amino acids at the N-terminus, RCAN1.1 is highly expressed in the nervous tissue, and the expression of RCAN1.4 in the peripheral tissues such as muscle is higher. high. Although the two are expressed differently in each organization, they are functionally similar. Summary of invention
• the invention provides the use of a peptide or nucleic acid encoding the same for the preparation of a medicament for the treatment of a disease associated with an increase in NF-KB activity, wherein the peptide comprises (a) comprises SEQ ID NO: 3 Or an amino acid sequence comprising (b) the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway .
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a peptide or a nucleic acid encoding the same, and optionally a pharmaceutically acceptable carrier or excipient, wherein the peptide (a) comprises SEQ ID NO An amino acid sequence of 3; or an amino acid sequence comprising (b) the amino acid sequence of SEQ ID NO: 3 modified, for example, substituted, deleted or added with one or more amino acids Column, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
• the invention provides a method of treating a disease associated with an increase in NF- ⁇ activity, comprising administering a peptide or a nucleic acid encoding the same to a patient having the disease, wherein the peptide (a) comprises SEQ ID NO: An amino acid sequence comprising 3; or (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
• the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) the amino acid of SEQ ID NO: Sequence composition, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory NF- ⁇ signaling pathway Or (e) consisting of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of (a) - (c), wherein The peptide has the effect of inhibiting the NF- ⁇ signaling pathway.
• the disease associated with increased NF- ⁇ activity is selected from the group consisting of: multiple myeloma, leukemia, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, Hodgkin Lymphoma, myelodysplastic syndrome, breast cancer, ovarian cancer, lung cancer, cervical cancer, prostate cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, throat cancer, cholangiocarcinoma, thyroid cancer, parathyroid cancer, and skin and neck Squamous cell carcinoma.
• the disease is preferably leukemia, for example selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, adult T-cell leukemia.
• leukemia for example selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, adult T-cell leukemia.
• FIG. 1 RCAN1 reduces nuclear transfer of NF- ⁇ and transcriptional activity of NF- ⁇ .
• A HEK293 cells were transfected with the RCAN1 expression vector pcDNA3.1 (-) RCANl.l-myc and pcDNA3.1 (-) RCAN 1.4-myc, and the gene knockout vector si-RCAN 1 and cells were harvested 48 hours later. The nuclei were isolated and lysed, subjected to SDS-PAGE, western blot using anti-NF- ⁇ antibody, and TBP (TATA binding protein) detected by anti-TBP antibody as internal reference of nuclear protein.
• TBP TATA binding protein
• the vector pNF-KBluc was co-transfected into HEK293 cells, and luciferase activity was measured 24 hours later using a luciferase kit to indicate the transcriptional activity of NF- ⁇ in cells. *: indicates statistical significance, p ⁇ 0.05. Control: empty vector.
• FIG. 2 Co-immunoprecipitation shows the presence of protein-protein interactions between RCAN1 and 1KB protein.
• HEK293 cells were transfected with the expression vector pcDNA3.1 (-) RC AN 1.1 -myc of RCAN1, and cells were harvested and lysed 48 hours later, (A) immunoprecipitation using anti-myc antibody (9E10), using anti- ⁇ antibody Western blot; (B) Immunoprecipitation using anti- ⁇ antibody, western blot using anti-myc antibody (9E10). Input for cell division before immunoprecipitation
• FIG. 3 Low expression of RCAN1 resulted in a significant increase in tyrosine phosphorylation of 1KB at position 42.
• HEK293 cells were transfected with the gene knockout vector si-RCAN1, cells were harvested 48 h later and lysed,
• A anti- ⁇ antibody was used Immunoprecipitation was performed using ⁇ 42 tyrosine phosphorylated antibody for western blot;
• B Immunoprecipitation was performed using IKB42 tyrosine phosphorylated antibody, and western blot was performed using anti- ⁇ .
• * indicates statistical significance, p ⁇ 0.05. Control: empty vector.
• FIG. 4 High expression of RCAN1 leads to death of acute leukemia cell lines as well as primary acute leukemia cells.
• the acute lymphoblastic leukemia cell line (A) and the acute myeloid lymphocytic leukemia cell line (B) were infected with the adenovirus of RCAN1, and the cell viability was measured 48 hours later using the cell viability kit.
• C Mononuclear cells were isolated from peripheral blood of leukemia patients and remission patients and control normal populations, and primary cultured leukemia cells were infected with adenovirus of RCAN1, and cell viability was measured 48 hours later using the cell viability kit.
• Av-GFP is an adenovirus expressing green fluorescent protein
• ⁇ -RCANl.l is an adenovirus expressing RCAN1.1 protein
• Av-RCANl.4 is expressed Adenovirus of RCAN1.4 protein (Ad-RCAN1.4 vector described in Example 1.2.2).
• Figure 5 The N-terminal 103 amino acids of RCAN1 bind to 1 KB and inhibit the transcriptional activity of NF-KB and kill acute leukemia cells.
• A HEK293 cells were transfected with the RCAN1 N-terminal 103 amino acid expression vector pcDNA3.1RCANl-103mychis or
• B the 103-197 amino acid pcDNA3.1RCAN103-197myc vector. After 48 hours, the cells were harvested and lysed. The -myc antibody (9E10) was immunoprecipitated and subjected to western blot using an anti- ⁇ antibody.
• the Jurkat leukemia cell line was transfected with the RCAN1 N-terminal 103 amino acid expression vector pcDNA3.1RCANl-103mychis or the 103-197 amino acid pcDNA3.1RCAN103-197myc plasmid, and the cells were assayed using the cell viability kit 48 hours later. Survival rate. * : indicates statistical significance, p ⁇ 0.05. Control: empty vector.
• FIG. 6 Purified TAT-RCANl-103 polypeptide can cause leukemia cell death.
• A SDS-PAGE shows that the purified TAT-RCANl-103 polypeptide is a single band.
• B TAT-RCANl-103 polypeptide was added to Jurkat cells, and cell viability was measured 48 hours later using a cell viability kit. *: indicates statistical significance, p ⁇ 0.05. Control: empty vector.
• regulation of the NF- ⁇ signaling pathway in vivo is mainly dependent on the 1KB of ⁇ inhibitory factor.
• Serine phosphorylation of 1KB at positions 32 and 36 promotes degradation of the proteasome by 1KB, and exposure to NF- after 1KB degradation.
• the nuclear localization sequence of the ⁇ molecule which is subsequently transferred into the nucleus, binds to the ⁇ response element on its target gene to regulate transcription of the target gene (Alkalay et al., 1995).
• the present inventors have found that the cleavage isomers RCAN1.1 (SEQ ID NO: 1) and RCAN1.4 (SEQ ID NO: 2) of the RCAN1 protein inhibit the NF- ⁇ signaling pathway and reduce the survival of cancer cells; further, The present inventors have found that a peptide having an N-terminal 103 amino acid of RCAN1.1 (SEQ ID NO: 3) can interact with 1 KB and inhibit NF- ⁇ activity, thereby reducing cancer The survival of the cell. Therefore, the inventors have for the first time revealed that the RCAN1 protein is an endogenous regulatory protein of NF- ⁇ , which can affect the NF- ⁇ signaling pathway, which interacts with the NF- ⁇ inhibitor 1KB and affects its tyrosine at position 42. Phosphorylation.
• the present invention provides the use of a peptide or a nucleic acid encoding the same for the preparation of a medicament for treating a disease associated with an increase in NF- ⁇ activity, wherein the peptide (a) comprises SEQ ID NO: Or an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
• the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO An amino acid sequence consisting of 3, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory factor of NF
• a sequence composition in which the peptide has an effect of inhibiting the NF- ⁇ signaling pathway is consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO
• peptide refers to a polymer of amino acid residues comprising two or more amino acids joined by peptide bonds.
• the polymer may be linear, sub-technical or cyclic, and may comprise naturally occurring and/or amino acid analogs which may be interrupted by non-amino acids.
• amino acid polymer is long (e.g., more than 50 amino acid residues), it is preferably referred to as a polypeptide or protein, but if it is 50 amino acids long or shorter, it is preferably referred to as a "peptide.”
• peptides described herein can be prepared by any suitable prior art methods, such as chemical synthesis, recombinant expression, and the like. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)) and Blackwell Scientific Publications, Nicholson, "Peptide Synthesis", Atherton And Chapter 9 of Sheppard "Peptide Synthesis”.
• the "modification" is preferably a conservative sequence modification well known in the art, including amino acid substitutions, additions or deletions.
• Amino acid modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis, and random PCR-mediated mutagenesis in nucleic acids encoding proteins.
• Conservative amino acid substitutions include those in which the amino acid residues are substituted by amino acid residues having similar structural or chemical properties. A family of amino acid residues having similar side chains has been identified in the art.
• amino acids with basic side chains eg lysine, arginine, histidine
• acidic side chain amino acids eg aspartic acid, glutamic acid
• uncharged polar side chain amino acids eg asparagine, glutamine, serine, threonine, tyrosine, semi-acid, isoleucine, valine, phenylalanine, methionine
• branched side chain amino acids eg Threonine
• Guidance on determining which amino acid residues can be substituted, inserted or deleted without eliminating their immunological activity can be found using computer programs well known in the art.
• Computer algorithms known to those skilled in the art such as Gap or Bestfit, can be used to optimize the comparison of amino acid sequences to compare and define similar or identical.
• substitution of one or more amino acids herein above can be in SEQ ID NO: 1, 2 or 3.
• Amino acid residues are substituted with amino acid residues having similar structural or chemical properties, such as mutual substitution between arginine proline, leucine and isoleucine.
• one or more amino acids deleted herein above may be an amino acid residue lacking position 1 of SEQ ID NO: 1, an amino acid residue at position 1 of SEQ ID NO: 2, or a ninth of SEQ ID NO: Amino acid residue at the position.
• the above-described addition of one or more amino acids may be the addition of the amino acid residue Ala at position 1 of SEQ ID NO: 1, the addition of amino acid residue Ala at position 1 of SEQ ID NO: 2, or the SEQ ID NO:
• the amino acid residue Ala is added at the 9th position in 3.
• inhibiting NF- ⁇ signaling pathway refers to inhibition of NF- ⁇ as a transcription factor. The activity of its downstream target gene.
• identity refers to the percentage of identical amino acids at the same or similar positions in the two protein chains.
• the BLAST algorithm was used to calculate the percent homology using the BLAST algorithm, which is available on the NCBI website (http://www.ncbi.nlm.nih.gov/).
• the peptides having sequence identity are functional homologs, i.e., they exhibit significant identity (e.g., about 50% sequence identity at the amino acid level) and perform the same function as their corresponding proteins.
• At least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more have the same sequence Sexual functional homologs are preferred functional homologs.
• nucleic acid As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” are used interchangeably to define a polymer of any length, such as a polydeoxyribonucleotide (DNA X eg eg cDNA, genomic DNA, plasmid, vector, isolated DNA and any mixture thereof) or polyribonucleotide (RNA) molecules (eg mRNA) or mixed polyribose-polydeoxyribonucleotides. They contain single or double stranded, linear or circular, natural or synthetic polynucleotides.
• DNA X polydeoxyribonucleotide
• RNA polyribonucleotide
• mRNA mixed polyribose-polydeoxyribonucleotides
• encoded refers to information that is included for translation into a given protein.
• the nucleic acid encoding the protein may comprise a non-translated sequence (e.g., an intron) within the translational region of the nucleic acid, or may lack such an inserted non-translated sequence (e.g., in a cDNA).
• the information encoding the protein is specified by the use of a codon.
• the nucleic acid of the invention may also be contained in a vector such as an expression vector, plasmid, virus, phagemid, phage, cosmid or artificial chromosome.
• a vector such as an expression vector, plasmid, virus, phagemid, phage, cosmid or artificial chromosome.
• an "expression vector” is a recombinant or synthetically produced nucleic acid construct having a series of designated nucleic acid elements that permit transcription of a particular nucleic acid in a host cell, eg, an expression vector other than the nucleic acid sequence to be expressed.
• a selection marker derived from a replication and control sequence compatible with the host for expression and a selectable phenotype conferring the transfected cell can also be included.
• the nucleic acid of the present invention can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment.
• suitable expression vectors are known in the art and are commercially available, such as prokaryotic expression vectors, yeast expression vectors, mammalian expression vectors, insect cell expression vectors, plant cell expression vectors.
• the nucleic acid sequence of the invention (SEQ ID NO: 1, 2 or 3) is in an expression vector such as pcDNA3.1 ( - ) mychis ( c ) ( Invitrogen ), pSuper ( Oligoengine ) or Adeno-X (Clontech ) Expressed in.
• “Host cell” refers to a cell that contains a vector and supports replication and/or expression of the vector.
• the host cell may be a prokaryotic cell such as an E. coli cell, or a eukaryotic cell such as a yeast cell, an insect cell, an amphibian cell or a mammalian cell.
• the host cell expressing the protein described herein may be any eukaryotic or prokaryotic cell used in the art, such as HEK293, HEK293T, E. coli cells, and the like.
• Various cell cultures as well as techniques for expression, collection, purification, and detection of expressed proteins are known to those skilled in the art. See, for example, J.
• the present invention also provides the use of a peptide or a nucleic acid encoding the same as a medicament for treating a disease associated with an increase in NF- ⁇ activity, wherein the peptide (a) comprises the amino acid sequence of SEQ ID NO: Or (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
• the present invention provides a pharmaceutical composition for treating a disease associated with an increase in NF- ⁇ activity, comprising a peptide or a nucleic acid encoding the same, and optionally a pharmaceutically acceptable carrier Or an excipient, wherein the peptide (a) comprises the amino acid sequence of SEQ ID NO: 3; or (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids. Wherein the peptide has the effect of inhibiting the NF- ⁇ signaling pathway.
• the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) the amino acid of SEQ ID NO: Sequence composition, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory NF- ⁇ signaling pathway
• the effect, or (e) is at least 95%, at least 96%, at least 97%, at least 98% or from the amino acid sequence in (a) - (c) Amino acid sequence consisting of at least 99% sequence identity, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
• the present invention provides a method of treating a disease associated with an increase in NF- ⁇ activity, comprising administering a peptide or a nucleic acid encoding the same or a pharmaceutical composition comprising the same to a patient having the disease
• the peptide (a) comprises the amino acid sequence of SEQ ID NO: 3; or (b) an amino acid comprising the amino acid sequence of SEQ ID NO: 3, such as substituted, deleted or added one or more amino acids.
• a sequence wherein the peptide has an effect of inhibiting the NF-KB signaling pathway.
• administering means that a predetermined amount of a substance is introduced into a patient by a suitable method.
• the isolated peptide, isolated nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition or vaccine of the present invention can be administered by any usual route as long as it can reach the desired tissue.
• a variety of modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, oral, topical, intranasal, intrapulmonary, and rectal, although the invention is not limited to these exemplified modes of administration.
• the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO An amino acid sequence consisting of 3, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory factor of NF
• a sequence composition in which the peptide has an effect of inhibiting the NF- ⁇ signaling pathway is consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO
• the peptides of the invention may also be fused to additional peptides to modify the physiological or pharmaceutical properties of the peptide, such as increasing stability or increasing membrane penetration.
• the peptide of the invention eg, SEQ ID NO: 3
• the human immunodeficiency virus HIV Tat protein eg, The 47-57 amino acid RKKRRQRRRG (SEQ ID NO: 14) is fused to facilitate penetration of the polypeptide into the cell membrane.
• the disease associated with an increase in NF- ⁇ activity refers to any disease associated with the occurrence, development, persistence or recurrence of the disease associated with an increase in NF- ⁇ activity (Basseres and Baldwin, 2006).
• the disease may be selected from the group consisting of multiple myeloma, leukemia, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome, breast cancer, ovarian cancer.
• the disease is preferably leukemia, for example selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, adult T-cell leukemia.
• treatment refers to: alleviating, alleviating, eliminating, obstructing, preventing the symptoms of the disease and/or delaying the onset and/or progression of the disease. Monitoring and/or measurement of the symptoms of the disease can be performed by the clinician using any means or method.
• Whether a peptide or a nucleic acid encoding the same can inhibit the activity of NF- ⁇ can be determined by measuring the amount of NF- ⁇ protein in the nucleus.
• a number of assays for determining NF- ⁇ protein are known in the art, such as immunoblotting of nucleoproteins, electrophoretic mobility experiments using isolated nuclear proteins, and assays for the activity of NF- ⁇ protein as a transcription factor using a reporter gene system (Imbert et al ., 1996).
• the amount of NF- ⁇ protein in the nucleus is determined by Western blotting to investigate the inhibitory effect of RCAN 1 on NF- ⁇ activity.
• leukemia is known to be a disease associated with an increase in the activity of NF- ⁇ .
• Leukemia is a blood-like tumor, and leukemia cell lines such as JURKAT are good leukemia cell models, especially short-term cultured primary leukemia cells, which are directly used for drug testing after isolation from peripheral blood of patients, cell survival based cytology.
• the method of measurement can reflect the effects and mechanisms of the drug.
• leukemia cell lines and primary cultured leukemia cells are used as models to study the effect of RCAN1 in the treatment of leukemia, and this model can prove that RCAN1 is a good candidate for treating leukemia.
• RCAN1 protein of the present invention or the N-terminal 103 amino acid fragment thereof is provided as a novel
• PCR amplified enzyme and dNTP purchased from Takarra;
• PCR amplification primers synthesized by Shanghai Shenggong Company;
• Takara T4 ligase purchased from Dalian Bao Bio Company;
• Adenovirus expression vector Adeno-X purchased from Clontech;
• E. coli DH5a was purchased from Invitrogen.
• HEK293 cell line was purchased from ATCC. HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum at a culture environment of 37. C constant temperature, 5% C0 2 cell culture incubator. Cell culture products were purchased from Invitrogen.
• Plasmid construction To construct the RCAN1 high expression vector, construct the pcDNA3.1 (-) RCANl.l-myc recombinant plasmid, and use the following primers to PCR-amplify the human cDNA library, and digest it with EcoR 1 and Kpn l and connect it with the same The vector was obtained by digesting the pcDNA3.1 (-) mychis (c) vector.
• the PCR primers used to clone RCAN1.1 are:
• Upstream primer ccgCTCGAGgccaccATGGAGGAGG TGGACCTGCA (SEQ ID NO: 4)
• a human cDNA library purchased from Clontech
• the above primers were used for conventional PCR (PCR conditions: 94 ° C lOmin, 30 cycles 94 ° C, 30 s, 55 ° C, 30 s, 72 ° C, 30 s, Finally, the gene fragment was amplified at 72 ° C for 5 min, and then the PCR amplified fragment was isolated and recovered by agarose gel electrophoresis, with the EcoR1 restriction site upstream and the Kpn l restriction site downstream.
• the DNA fragment obtained by PCR was digested with EcoR 1+Kpn I and ligated into pcDNA3.1 (-) mychis (c) vector which was also digested with EcoR 1+Kpn I to obtain pcDNA3.1 (-) RCANl. L-myc recombinant expression plasmid.
• the PCR primers used to clone RCAN1.4 are:
• Upstream primer ccgCTCGAGgccaccATGCATTTTA GAAACTTTAA ( SEQ ID NO: 6 )
• Downstream primer atggtacc CCTCTTCTTCCTCCTTC ( SEQ ID NO: 7 )
• Ad-RCAN1 .1 and Ad-RCANl .4 vectors The pRCAN1.E-EGFP and pRCANl.4-EGFP vectors were digested with Nhe I and Not I, and a fragment of about 1.4 kb was recovered as an insert and inserted into the same restriction enzyme.
• pShuttle2 purchased from Clontech
• pShuttle2-RCAN 1.1 -EGFP and pShuttle2-RCAN 1.4-EGFP was obtained by pShuttle2-RCAN 1.1 -EGFP and pShuttle2-RCAN 1.4-EGFP, and then pShuttle2-RCANl.l-EGFP and pShuttle2-RCAN1.4-EGFP were digested with I-ceuI and Pl-Scel.
• a fragment of about 1.4 kb was inserted as an insert into the same restriction enzyme Adeno-X adenovirus vector (purchased from Clontech) to obtain Ad-RCAN1.1 and Ad-RCAN 1.4
• leukemia cell lines cannot be transfected using general transfection methods, we used adenovirus to express RCAN1 protein. About 106 HEK293 cells were seeded one day prior to transfection into 60mm culture dish, cells were transfected using liposome LF2000 (available from invitrogen) and 5 g of Pacl (available from NEB) digestion viral vector or Adeno-X Ad-RCANl.1 and Ad-RCAN 1.4 (see the instructions of Invitrogen's LF2000 Transfection Kit for specific methods). After 7 days, cells were harvested and 3 freeze-thaw lysed cells were used to collect virus-containing lysate.
• liposome LF2000 available from invitrogen
• Pacl available from NEB digestion viral vector
• Adeno-X Ad-RCANl.1 and Ad-RCAN 1.4 see the instructions of Invitrogen's LF2000 Transfection Kit for specific methods. After 7 days, cells were harvested and 3 freeze-thaw lysed cells were used to collect virus-containing lys
• HEK293 cells inoculated with 60 mm culture dishes were infected with 0.5 ml of cell lysate. After 48 hours, the cells were frozen and thawed to collect virus, and the same method was continued. The virus collected after infection with HEK293 twice was used for the experiment. The virus titer is approximately 4 X 10 8 /ml. In the present invention, the cell line and primary cells use a multiplicity of infection (MOI) of 30 unless otherwise specified.
• MOI multiplicity of infection
• HEK293 cells were transfected with RCAN1 expression vector pcDNA3.1 (-) RCANl.l-myc: HEK293 cells were transfected with liposome LF2000 (purchased from invitrogen), and 4 ⁇ l of LF2000 was added to opti-MEM of ⁇ (purchased from Invitrogen) After 5 minutes, it was mixed with 2 g of the RCAN1 expression vector pcDNA3.1 (-) RCANl.l-myc in opt ⁇ of opti-MEM, and then allowed to stand at room temperature for 15 minutes, and added to a 35 mm diameter culture dish containing HEK293. . After 48 hours, the nuclei were separated and lysed: The nuclei were separated and lysed using a nuclear protein extraction kit (purchased from Millipore).
• the nuclear lysate was separated using 12% glycine SDS-PAGE (purchased from Biorad): The gel and transfer device was Biorad's mini-protean 3 vertical electrophoresis device.
• NF- ⁇ in the nucleus of cells with high expression of RCAN1 was significantly decreased (see Figure 1), indicating that RCAN1 can significantly inhibit nuclear transfer of NF-KB, and RCAN1 knockdown can significantly increase nuclear transfer of NF-KB (Fig. 1A). .
• the RCAN1 expression vector was co-transfected with HEK293 cells with pNF-KB-luc (purchased from Clontech;) and pRL-TK (purchased from Promega) (the plasmid usage ratio was 1: 1 : 0.02, see Invitrogen LF2000 transfection for specific methods).
• the kit instructions lysing the cells 24 hours later, and measuring the fluorescence using a dual fluorescence reporter system (purchased from Promega).
• HEK293 cells were transfected with RCAN1 expression vector (as described in Example 2 2.1);
• the supernatant was centrifuged at 2000 g, and the pellet was washed twice with PBS, and the loading buffer was added; 12% glycine SDS-PAGE was used to separate the protein and the protein was transferred to the PVDF membrane; Western blot was used to detect RCAN1 protein using anti-myc antibody (purchased from ABcam) (see ABcam's antibody specification for specific methods).
• the protein can be precipitated using an anti-myc antibody, and a 1KB antibody can be used for western blot.
• the method of SDS-PAGE and western blot is specifically described in Example 2 2.1.
• RCAN1 affects 1KB tyrosine phosphorylation at position 42
• HEK293 cells were transfected with RCAN1 knockout vector si-RCAN1 (as described in Example 2 2.1, the amount of vector Si-RCAN1 was also 2ug);
• Leukemia cell lines Jurkat, NALM-6, CEM, MOLT-4, Kasumi, HL-60, THP-1, HEL were purchased from ATCC. Primary leukemia cells and cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and cell culture products were purchased from Invitrogen. The culture environment is 37. C constant temperature, cell culture incubator with 5% CO 2 .
• Cell viability kit Celltiter-Glo Luminescence Kit purchased from Promega.
• the primary leukemia cells were isolated using Histopaque reagent. 3 ml of Histopaque-1077 reagent was added to a 15 ml centrifuge tube, and the upper layer was 3 ml of fresh peripheral blood of leukemia patients. After centrifugation at 300 g for 30 minutes, the second layer of mononuclear cells was aspirated and washed once with 10 ml of PBS buffer, and inoculated into the culture dish.
• Leukemia cell lines or primary leukemia cells were infected with adenovirus expressing RCAN1.1 and RCAN1.4 for 48 hours, and cell viability was measured using a cell viability kit.
• pcDNA3.1RCAN103-197myc plasmid Using primers DSCR1-103F: ccgCTCGAGgccaccatg C AGTTTCTGAT CTCCCCT (SEQ ID NO: 12) and reverse primer BGH (TAGAAGGCACAGTCGAGG (SEQ ID NO: 13)), pcDNA3.1 (-) RCANl.l -myc was used to amplify the 103-197 fragment of the substrate, and the pcDNA3.1RCAN103-197myc recombinant plasmid was obtained by inserting the restriction enzyme sites EcoR 1 and Kpnl into pcDNA3.1 (-) mychis (c).
• Nuclear transfection kit VCA-1003 purchased from Lonza.
• Co-immunoprecipitation HEK293 cells were transfected with the vectors pcDNA3.1RCANl-103mychis and pcDNA3.1RCAN103-197myc expressing the N- or C-fragment of RCAN1, respectively. The co-immunoprecipitation experiment was carried out as in the experimental method shown in Example 3.
• NF- ⁇ activity fluorescent indicator system HEK293 cells were transfected with the vectors pcDNA3.1 RCAN 1 - 103mychis and pcDNA3.1 RCAN 103-197myc expressing the N- or C-fragment of RCAN1, respectively, using the method as in Example 2. NF- ⁇ activity was examined.
• the TAT sequence is the 47th to 57th amino acids of the human immunodeficiency virus HIV Tat protein (RKKRRQRRRG (SEQ ID NO: 14)), and this TAT sequence is fused to the N-terminal polypeptide of RCAN1 to promote The polypeptide penetrates the cell membrane.
• the coding oligonucleotide Tatg agg aagaagcggagacagcgacgaaga ggatcc c (SEQ ID NO: 15) containing the TAT fragment was inserted into the prokaryotic expression of pet-28b digested with Nde I and Xho I restriction enzymes (purchased from New England Biolabs).
• the vector (purchased from Novagen) was used to obtain the pet-28bTAT vector, then the pet-28bTAT vector was digested with Bamh l and Xho I, and the PCR fragment of RCAN1 1-103 was inserted, PCR upstream primer: CGGGATCCATGGAGGAGGTGGACCTG (SEQ ID NO: 16); downstream Primer: CCGCTCGAGCTTGTCTGGATTTGGCGGA (SEQ ID NO: 17).
• the PCR template was pcDNA3.1 (-) RCANl.l-myc expression vector.
• RCAN1 N-103 polypeptide 50 ng of pTAT-RCAN1N-103 expression vector was transformed into 21 ⁇ BL21(DE3)pLysS cells (purchased from Promega;) by ice cream for 30 minutes, heat shock for 40 seconds, iced 5 minute.
• the clone expressing TAT-RCAN1N-103 was inoculated into 200 ml of LB medium containing 100 g/ml of ampicillin, shaken at 225 rpm for 16 hours at 37 ° C, and inoculated again to 1 liter of LB culture containing 100 g/ml of ampicillin. Base, shake at 37 ° C overnight.
• the cell pellet was collected by centrifugation at 5000 g for 10 minutes, washed once with PBS, and the cell pellet was dissolved in 20 ml of cell suspension (containing 8 M urea, 100 mM sodium chloride, 20 mM HEPES, pH 8.0), and subjected to 3 times of 15 seconds of ultrasound.
• the cells were pulverized, 16000 g, centrifuged at 4 ° C for 15 minutes, and the supernatant was collected, and the affinity chromatography column HisTrapTM (purchased from amersham pharmacia) was passed. Proteins were resolved from the column using a cell suspension containing 500 mM imidazole, demineralized using a PD-10 column (purchased from GE). Salt was stored at -80 ° C in PBS containing 10% glycerol (purchased from invitrogen) ).
• Jurkat cells were treated with the purified TAT-RCAN1N-103 polypeptide: Jurkat cells were treated with ⁇ TAT-RCAN1N-103 polypeptide, and cell viability was measured using MTT 24 hours later. The results showed that Jurkat cells were compared in the TAT-RCAN1N-103 treated group. The survival rate of the normal control group was significantly reduced (Fig. 6B).
• Transcription factor NF-kappaB is constitutively activated in acute lymphoblastic leukemia cells. Leukemia 14, 399-402.
• Epidermal growth factor activates nuclear factor-kappaB through IkappaBalpha kinase-independent but EGF receptor-kinase dependent tyrosine 42 phosphorylation of IkappaBalpha.
• Oncogene 26 , 7324-7332.

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